The human-specific pathogen, Neisseria gonorrhoeae is responsible for the sexually-transmitted infection, gonorrhea. This disease is common among both men and women, but causes significant morbidity amongst women. During the course of infection, N. gonorrhoeae must obtain a necessary nutrient, iron, from the human host. This pathogen has evolved the capacity to assimilate iron from a variety of host-specific sources, including from the iron-binding glycoproteins, transferrin and lactoferrin. Iron transport from these iron-binding proteins is accomplished by an integral, outer membrane transporter, which requires energy and the TonB protein. Because the TonB-dependent transporters share sequence homology, genes that encode them can be identified within sequenced bacterial genomes. The presence of genes in the N. gonorrhoeae genome suggests that other nutrients can be internalized via these uncharacterized transporters and that these nutrients, potentially including iron, may be crucial for gonococcal survival within the human host. Some iron chelates are internalized by N. gonorrhoeae in an energy- and TonB-independent pathway, suggesting that the gonococcus has multiple, overlapping and potentially redundant iron acquisition systems, which emphasizes the importance of obtaining this nutrient. The long-term goal of this research is to identify functional, TonB- dependent or TonB-independent transport systems, to characterize their ligands, to probe their mechanism of uptake, and to determine the biological relevance of interference with transporter function in vivo. These studies are significant because components of these pathways could ultimately be used as vaccine antigens for immunoprophylaxis or as targets for therapeutics to treat gonococcal disease. The overall goal of the proposed study is to determine how TonB-dependent and TonB-independent transport processes contribute to the ability of N. gonorrhoeae to grow in vitro and within cultured host cells. The specific aims of this proposal address the following questions: 1. By what molecular mechanism does the gonococcus acquire iron from low molecular iron-chelates produced by other bacteria? 2. What function does the host-cell expressed TonB- dependent transporter, TdfF, serve in intra-epithelial cell gonococcal survival? The methods to be employed to address these questions include directed and random mutagenesis to identify key components of the TonB- independent iron-chelate utilization pathway. The mechanisms that control expression of the TonB-dependent transporter, TdfF will be evaluated by genetic fusions, RT-PCR and protein expression profiling. Finally, the contribution that TdfF provides to growth and survival within cultured cells will be evaluated using intracellular survival assays and state-of-the-art immunofluorescence microscopy techniques.